Ongoing advances in nucleic acid (DNA and RNA) molecular biology have greatly contributed to the growth of knowledge in all fields of biology. The entire body of traditional disciplines in biology has benefited from this molecular biology contribution. Mention may be made of microbiology and its branches: bacteriology, mycology, parasitology and virology, but also of cellular biology, cellular physiology, molecular genetics, biochemistry, enzymology, immunology and animal physiology. Use of the potentials of nucleic acids has resulted in the appearance of new, hitherto unknown fields of investigation known by new terms such as genomics, molecular medicine, and gene therapy.
Recombinant DNA technologies have grown just as rapidly thanks to the use of the bacterium Escherichia coli. The K12 strain of Escherichia coli together with its many mutants has, over the years, become an essential intermediary in all operations involving nucleic acids. Any researcher using recombinant DNA in almost all fields of biology is led to work with this bacterium, particularly in the form known as transformed, as a result of the introduction of plasmid or viral DNA into the cell. This is how, for example, the great majority of recombinant DNA preparations, which may include fragments of natural or modified DNAs of different origins obtained from the realm of procaryotic and eucaryotic, archeons or even chemically synthesized, are made from vectors specific to Escherichia coli. Laboratory strains of the yeast Saccharomyces cerevisiae must also be mentioned as it is one of the most frequently used microorganisms for the production of recombinant DNA.
The transformation of a bacterium or of a eucaryotic microorganism by a vector is an operation that is frequently performed by researchers and is performed in two stages. The first stage consists in introducing the vector""s DNA into cells by means of various techniques, and the second stage in selecting those cells in the population that have received and express the vector""s genes. The proportion of cells that express the vector""s genes among the total number of cells varies greatly according to the type of transforming DNA and the transfer procedure used. Even in the best cases the population of transformed cells is always small. Several methods have therefore been devised to select only those cells that express the new gene or genes introduced by the transforming DNA. All rely on the expression of a gene whose product gives a dominant character to the cell relative to non transformed cells. An example is the use of a gene that is resistant to an antibiotic that is normally toxic to the receiving microorganism. The addition of a given antibiotic to a microbial cell that has been in contact with the DNA carrying the gene that is resistant to the antibiotic either prevents growth or kills cells that do not express the transgene, according to the nature of the antibiotic, whereas on the contrary the transformed cells that have become resistant multiply to finally produce a pure culture of resistant cells. For practical reasons selection is usually done with solid media in a Petri dish. In this case each transformed cell produces an independent pure culture that appears as the formation of colonies on the surface of the medium contained in the Petri dish. The most frequently used antibiotics for selecting transformed clones in E. coli are ampicillin and carbenicillin among the penicillins, kanamycin and neomycin among the aminosides, tetracyclin, chloramphenicol and zeocin.
The use of selection markers is often associated with the use of identification markers, in other words genes that enable transformed colonies to be distinguished from colonies that do not express said transgene by a different color. The most popular system among researchers is that based on the expression of the lacZ gene of the Escherichia coli xcex2-galactosidase. The action of this enzyme, either whole or reconstituted as in the alpha peptide system used in many of the cloning vectors in E. coli leads to hydrolysis of a colorless compound, 5bromo-4-chloro-3-indoxyl-xcex2-D-galactopyranoside (or X-gal) to give a blue insoluble product. The presence of this coloring agent in solid media for E. coli, for example, leads to the appearance of blue colonies formed by cells producing an active xcex2-galactosidase, whereas cells devoid of xcex2-galactosidase produce white colonies. Many other chromogenic or fluorescent substrates of xcex2-galactosidase are used for the same purpose of visual distinction of positive populations among a population of white or non fluorescent colonies. Similar chromogenic or fluorescent substrates are also often used to detect positive colonies for other enzymatic activities such as xcex2-glucuronidase and alkaline phosphatase, for example.
Gene cloning in E. coli vectors no longer requires a level of qualification needing several years experience as was still the case in the 1980s. Acquisition of these standard technologies often begins at school and is carried on in molecular biology courses at university in any country with a strong scientific tradition. Methodologies relating to DNA and RNA manipulation have been greatly simplified by the introduction of successive improvements designed by the entire scientific community and propagated through publications. Another decisive element that has led to simplification and also to reduction of the time needed to perform experiments relating to DNA and RNA has come from companies specialized in the supply of products for research into and use of microbiology and molecular biology. By supplying equipment that is ready-to-use and contains all the elements needed to perform even the most complex experimental operations in biology these companies have greatly contributed to make these technologies more accessible. These companies readily impart information on any technological improvement discovered in the world""s scientific and medical laboratories to their biologist clients. To date none of them offer the products and the methodology that are the subject of this invention.
The different stages in the transformation of a bacterium or of eucaryotic microorganisms such as yeasts and filamentous fungi by a DNA vector are broadly as follows: recipient cells enabled to incorporate DNA by means of various treatments are put into contact with the DNA and are then put into culture in a liquid medium to allow the appearance of the selection property before being spread in Petri dishes containing a solid medium with the addition of the selection agent. At this stage a chromogenic or fluorescent substrate may also be added for the cloning or transfer vectors as a means to identify the colonies. Following one night or several days incubation, according to the growth rate of the organism concerned, those cells that initially recovered the vector and express the transgenes form colonies. These colonies are then recovered in order to prepare cultures, this time in liquid media containing the selection agent, in order to extract the extrachromosomal DNA which is then characterized on the basis of size, the presence of restriction enzyme sites, and possibly by sequencing. The volume of liquid culture depends on the quantity of DNA sought by the researcher, and may vary from a few milliliters to one liter or more. These technologies, as applied for instance to microorganisms such as E. coli and S. cerevisiae, are well known to those familiar with the art, and their degree of difficulty in no way limits implementation of the invention.
The invention relates to a new method of preparing selective liquid and solid media for the selection and manipulation of recombinant microorganisms. The selective media are at present prepared in an identical way by all experimental biologists all over the world. The ingredients that compose a given medium are mixed with water in variously shaped containers usually made of glass and closed either with a cork made of cotton, polyurethane foam, or any other matter permeable to air, able to withstand temperatures of 125-130xc2x0 C. and wrapped in aluminum foil or closed with a screw cap made of bakelite or other resistant material. The container is then placed in an autoclave for sterilization, generally for 20 minutes at 120xc2x0 C. After autoclaving the container is cooled to a temperature of 40-50xc2x0 C. and only then is the antibiotic in a sterile solution, as well possibly as a sterile solution of a chromogenic or fluorescent substrate and an inductive molecule of the expression of a transgene, added aseptically to the medium in the container. These various additives cannot be included in the medium before autoclaving owing to their sensitivity to the temperature reached in the autoclave, and are therefore only added in the form of a sterile solution by filtering through a membrane in the medium following autoclaving. In the case of a medium containing agar the dishes are immediately prepared by spreading the melted medium in sterile Petri dishes.
The three stages of the sterilization cycle in the autoclave, i.e. the rise in temperature, the duration of maintenance at the desired temperature, and the fall in temperature, takes at least one hour. Performance of the other tasks involved in the process, that is preparation of the medium before autoclaving, cooling of the medium following opening of the door of the autoclave, addition of the additives and preparation of the Petri dishes takes at least another hour, bringing the total time of the operation to about two hours. If the problem of availability of the autoclave is added, a bulky apparatus which may be the only one in a laboratory, and which may not always be available for use at the time a researcher needs it most, and the cause of omissions or errors arising in the additions of antibiotics and other molecules to the medium, a process is achieved which so far has been necessary but represents a serious limitation to greater efficiency in the researcher""s working time. A first solution at this level is the centralization of preparation of the media by one or a group of persons who ensure the supply of the culture media to a whole group of users. This situation can of course only exist in large units. A second possibility is the purchase of liquid media ready for use and Petri dishes ready for use, a solution which would be perfect were it not for the cost, a limiting factor for the budgets of many laboratories and teams both in the public and private sectors.
The use of microwave ovens has become general practice for the heating of food kept either at room temperature or refrigerated or deep frozen. The advantages of microwave ovens over traditional methods of heating food lie in the speed and ease of implementation. The high demand in the market has led to microwave ovens being so commonplace as to be as common in kitchens as refrigerators. A great variety of ovens, differing in wave strength, internal volume and programming options is now available on the domestic appliances market. Microwave ovens have also in past years found a place in laboratories for specific applications in molecular biology. Characterization of the size of nucleic acids is done by measuring migration distances following electrophoresis in agarose gel. Agarose, which is the solidifying agent of the gel, is a purified form of the gelose used to solidify culture media. A now widely used technique consists in melting the agarose with the addition of the aqueous buffer in a microwave oven in order to be able to pour the liquid solution into the vat of the electrophoresis apparatus to form the gel. Sterility of the gel is entirely unnecessary in this application, and only speed has led to the generalization of this technique as opposed to that which relies on fusion of the agar in an autoclave.
It would be very advantageous to be able to substitute the use of an autoclave by that of a microwave oven for the preparation of liquid and solid culture media. However the essential condition here is that the media must be completely sterile, in other words devoid of microorganisms. It is because microwave ovens have always been considered unable to ensure sufficient sterility that they have not been recommended for the preparation of culture media for microorganisms. Confirmation of this assertion is to be found in the basic reference book of every microbiologist (Handbook of Microbiological Media, second edition, 1997, by Ronald M. Atlas, Edited by Laurence C. Parks, CRC Press), which provides references to thousands of media, including their compositions and methods of preparation, but does not once mention the use of microwave ovens instead of autoclaves.
The effect of microwaves applied for periods compatible with the desired use is not sufficiently microbiocidal to kill all microorganisms present in the solid ingredients of media. Ingredients of media consisting of organic matter of animal, plant or microbial origin and mineral matter contain at different levels a great diversity of microorganisms.
One of the methods that make it possible to obtain sterile media by microwaving is to sterilize the raw materials used in the composition of the media before exposure to microwaves. The powdered mixture of each of the constituents may be sterilized, for example, by gamma irradiation in equipment already in use in establishments equipped to sterilize food. Maintenance of sterility of irradiated powder before use is ensured by aseptic storage of the powder in sterile packets containing a unit quantity of powder sufficient to prepare a given quantity of medium. The manufacture of culture media that meet the required sterility criteria is thus achieved by using these packets in the following conditions: a sterile container closed by a non metallic screw cap in which the contents of a packet corresponding to a given medium have been poured and mixed with the required quantity of water is placed in a microwave oven. The container is subjected to the effects of microwaves for about three minutes at the oven""s maximum power with two or three regularly spaced pauses during which the container is agitated by rotation in order to facilitate a uniform dissolution of the powders, and the melting of the agar in the case of solid media. Following a 5 to 10 minute cooling period the corresponding antibiotic and other additives are added to produce a liquid medium ready for use or a solid medium ready to be poured for the manufacture of Petri dishes.
An even greater simplification of the preparation process of media by the use of microwaves would be achieved if the selection and identification agents were already present in the contents of the packet. A number of these additives have proven sensitive to temperatures generated by the microwaves and can therefore not be included in the powders.
However in the course of the great many trials made to perfect this new process, the applicant company has found that the constituents of the composition of some media played a thermoprotective role on additives sensitive to heat such as antibiotics of the penicillin type, or to X-gal, for example. Substances known to be cryoprotective and for their effect in protection during lyophilisation such as saccharose, trehalose, amylopectins, various starches as well as mannitol and sorbitol have proven in different degrees to increase the resistance of molecules to inactivation by heat generated by microwaves in aqueous solutions. Thanks to the addition of one of these stabilizing substances, the choice and concentration of which must take into account an absence of effect on the growth of the microorganism being studied, the present invention makes it possible to include most of the selection and identification additives of the recombinant microorganisms in the packets, thus eliminating a post microwave stage.